Transformation of hereditary material of plants

ABSTRACT

A novel process for the direct transfer of foreign genes to plant genomes is described. The novel process comprises placing a gene under the control of plant expression signals and transferring it, by contact with protoplasts without the aid of natural systems for infecting plants, direct to plant cells from which genetically transformed plants can subsequently be derived.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/473,893,filed Jun. 7, 1995 and issued Mar. 13, 2001 as U.S. Pat. No. 6,201,169,which is incorporated herein by reference and which is a continuation ofapplication Ser. No. 08/038,778, filed Mar. 29, 1993 and issued Sep. 26,1995 as U.S. Pat. No. 5,453,367, which is a continuation of Ser. No.07/485,790, filed Feb. 23, 1990 and issued Jul. 27, 1993 as U.S. Pat.No. 5,231,019, which is a continuation-in-part of abandoned applicationSer. No. 07/366,285, filed Jun. 13, 1989, which is a continuation ofabandoned application Ser. No. 07/157,115, filed Feb. 10, 1988, which isa continuation of abandoned application Ser. No. 06/730,025, filed May3, 1985.

FIELD OF THE INVENTION

The present invention relates to a novel process for transforminghereditary material of plants and to the plant products obtained by saidprocess.

Plants having novel and/or improved properties can be produced byintroducing new genetic information into plant material.

BACKGROUND

In view of the rapid rise in world population and the concomitantincrease in the need for food and raw materials, increasing the yield ofuseful plants as well as the increased extraction of plant storagesubstances, and in particular advances in the field of nutrition andmedicine, are among the most urgent tasks of biological research. Inthis connection, the following essential aspects may be mentioned by wayof example: strengthening the resistance of useful plants tounfavourable soil or climatic conditions as well as to disease andpests; increasing resistance to plant protective agents such asinsecticides, herbicides, fungicides and bactericides; and a usefulchange in the nutrient content or of the harvest yield of plants. Suchdesirable effects could be produced generally by induction or increasedformation of protective substances, valuable proteins or toxins. Acorresponding influence on the hereditary material of plants can bebrought about, for example, by inserting a specific foreign gene intoplant cells without utilising conventional breeding methods.

The transfer of novel DNA sequences into plant cells using geneticallymanipulated plant infecting bacteria has been described in theliterature in a number of publications, for example Nature, Vol. 303,209-213 (1983); Nature, Vol. 304, 184-187 (1983); Scientific American248(6), 50-59 (1983); EMBO-Journal 2(6), 987-995 (1983); Science 222,476-482 (1983); Science 223, 247-248 (1984); or Proc. Natl. Acad. Sci.USA 80, 4803-4807 (1983). In these publications, the natural propertiesof these bacteria for infecting plants were utilised to insert newgenetic material into plant cells. So far such insertion has been madeusing preferably Agrobacterium tumefaciens itself or the Ti plasmidthereof, and also cauliflower mosaic virus.

SUMMARY

In contradistinction thereto, the novel process of this invention makespossible the direct transfer of a gene without the use of biologicalvectors, in particular, without the T-DNA border regions of theTi-plasmid. Pathogens have been used as vectors in the known processes.As the process of this invention is performed without pathogens, thelimitations imposed by the host specificity of pathogens also do notapply. The development of the plants on which the novel process oftransformation is carried out is not impaired by said process.

In addition to the process for transforming hereditary material ofplants, the present invention also relates to the products obtainable bysaid process, in particular protoplasts and plant material derivedtherefrom, for example cells and tissues, in particular complete plantsthat have been regenerated from said protoplasts and the geneticallyidentical progeny thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the construction of the pABDI and pABDII plasmids.

FIG. 2 depicts the construction of the pCaMV6Km plasmid.

DETAILED DESCRIPTION

Within the scope of the present invention, the following definitionsapply:

gene: structural gene with flanking expression signals structural gene:protein-coding DNA sequence expression signals: promoter signal andtermination signal plant expression expression signal that functions inplants signal: promoter signal: signal that initiates transcriptiontermination signal: signal that terminates transcription enhancersignal: signal that promotes transcription replication signal: signalthat makes possible DNA replication integration signal: DNA sequencethat promotes the integration of the gene into genomic DNA hybrid gene:gene constructed from heterologous DNA sequences, i.e. DNA sequences ofdifferent origin that may be natural as well as synthetic DNA sequencescarrier DNA: a neutral (i.e. not participating in the function of thegene) DNA sequence flanking the gene isolated gene: DNA sequence codingfor a single protein and separated from the original DNA NPT II gene:neomycin 3′-phosphotransferase gene, type II, of transposon Tn 5[Rothstein, S. J. and W. S. Retznikoff, Cell 23, 191-199 (1981)] genomicDNA: DNA of the plant genome (total or part thereof).

The present invention is concerned with a novel process for thetransformation of hereditary material of plants, which process comprisestransferring a gene direct into plant cells without the aid of naturalsystems for infecting plants. Such transformation is accordingly avector-free transformation. In this vector-free transformation, theforeign gene for insertion is under the control of plant expressionsignals. The vector-free transformation of plant genes is preferablycarried out by introducing a foreign gene for insertion into plant cellstogether with plant protoplasts acting as recipients (receptorprotoplasts) into a suitable solution and leaving them until the genehas been taken up by the protoplasts.

As protoplasts it is preferred to use those of a single plant species orof a systematic unit which is a suborder of a species. The foreign geneand the protoplasts are conveniently left in the solution for a periodof time ranging from several seconds to several hours, preferably from10 to 60 minutes, most preferably for 30 minutes.

The process of this invention is susceptible of broad application. Thusit is possible to transfer any structural genes of plant origin, forexample the zein gene [Weinand, U., et al., Mol. Gen. Genet. 182,440-444 (1981)], of animal origin, for example the TPA gene [tissue-typeplasminogen activator gene; Pennica, D., et al., Nature 301, 214-221(1983)], of microbial origin, for example the NPT II gene, or also ofsynthetic origin, for example the insulin gene [Sepien, P., et al., Gene24, 289-297 (1983)], into hereditary material of plants, provided thatthe structural genes are flanked by expression signals which areexpressed in plants and which expression signals may be of plant,animal, microbial or synthetic origin.

The transferred genes, consisting of structural gene and flankingexpression signals, may be naturally occurring genes as well as hybridgenes. In the process of this invention, it is preferred to use thosegenes whose expression signals are of animal or, in particular, of plantor synthetic origin. Exemplary of such genes are:

a) complete genes of plants consisting of the structural gene with itsnatural expression signals;

b) completely synthetic genes consisting of a structural gene ofsynthetic origin, flanked by expression signals of synthetic origin;

c) structural genes of plant origin, flanked by plant expressionsignals, with the structures and expression signals originating fromvarious plant species;

d) structural genes of plant origin, flanked by expression signals ofsynthetic origin;

e) structural genes of animal, microbial or synthetic origin, flanked byexpression signals of plant origin; or

f) structural genes of animal or microbial origin, flanked by expressionsignals of synthetic origin.

Most preferred are structural genes of bacterial origin, flanked byexpression signals of plant origin, in particular those originating fromplant viruses. Particularly suitable expression signals for use in theprocess of this invention are the expression signals of gene VI ofcauliflower mosaic virus.

The hybrid genes are prepared by microbiological techniques which areknown per se, retaining the reading frame of the coding for the proteinsto be produced by the plant cell. Such techniques are known and aredescribed e.g. in the following publications: “Molecular Cloning”,Maniatis, T., Fritsch, E. F. and J. Sambrook, Cold Spring HarborLaboratory, 1982, and “Recombinant DNA Techniques”, Rodriguez, R. L. andR. C. Tait, Addison-Wesley Publishing Comp., London, Amsterdam, DonMills. Ontario, Sydney, Tokyo, 1983.

To integrate the foreign gene into the genomic DNA of the plant cell, itis advantageous if the gene, consisting of structural gene and plantexpression signals, is flanked by neutral DNA sequences carrier DNA).The carrier DNA may consist of two linear DNA strands, so that theconstruction to be inserted into the plant cell is a linear DNAmolecule. The DNA sequence prepared for the gene transformation can,however, also have an annular structure (plasmid structure). Suchplasmids consist of a DNA strand into which the foreign gene containingthe expression signals is integrated. The carrier DNA can be ofsynthetic origin or can be obtained from naturally occurring DNAsequences by treatment with suitable restriction enzymes. Thus, forexample, naturally occurring plasmids which have been opened with aselective restriction enzyme are suitable for use as carrier DNA.

Exemplary of such a plasmid is the readily obtainable pUC8 plasmid(described by Messing, J. and J. Vieira, Gene 19, 269-276, 1982).Fragments of naturally occurring plasmids can also be used as carrierDNA. For example, the deletion mutant for gene VI of cauliflower mosaicvirus can be used as carrier DNA.

The probability of the genetic transformation of a plant cell can beenhanced by different factors. Accordingly, as is known from experimentswith yeast, the number of successful stable gene transformationsincreases

1) with the increasing number of copies of the new genes per cell,

2) when a replication signal is combined with the new gene, and

3) when an integration signal is combined with the new gene.

The process of this invention is therefore susceptible of especiallyadvantageous application when the transferred gene is combined with areplication signal which is effective in plant cells or with anintegration signal which is effective in plant cells, or which iscombined with both signals.

The expression of a gene in a plant cell depends on the transcriptionfrequency of the gene in a messenger RNA sequence. It is thereforeadvantageous if the new gene is combined with an enhancer signal thatpromotes this transcription. Methods meriting particular attention arethose for transferring a gene which is combined with replication,integration and enhancer signals that are effective in plants.

It is further of great technical advantage if the transferred gene has aselective marker function, i.e. if the transformed plant cells can beseparated from the non-transformed plant cells under specific conditionsof selection. A marker function of this kind permits the process to becarried out efficiently in that only those plant cells need to beregenerated to calli or complete plants by microbiological techniques,the hereditary material of which plants contains a gene capable ofexpression that permits marker-specific methods of selection.

Whereas protoplasts, cell culture cells, cells in plant tissues, pollen,pollen tubes, egg-cells, embryo-sacs or zygotes amd embryos in differentstages of development are representative examples of plant cells whichare suitable starting materials for a transformation, protoplasts arepreferred on account of the possibility of using them direct withoutfurther pretreatments.

Isolated plant protoplasts, cells or tissues can be obtained by methodswhich are known per se or by methods analogous to known ones.

Isolated plant protoplasts which are also suitable starting materialsfor obtaining isolated cells and tissues can be obtained from any partsof the plant, for example from leaves, embryos, stems, blossoms, rootsor pollen. It is preferred to use leaf protoplasts. The isolatedprotoplasts can also be obtained from cell cultures. Methods ofisolating protoplasts are described e.g. in Gamborg, O. L. and Wetter,L. R., Plant Tissue Culture Methods, 1975, 11-21.

The transfer of the new genes into plant cells is effected directwithout using a natural system for infecting plants such as a plantbacterium, a plant virus, or transfer by insects or phytopathogenicfungi. This is achieved by treating the plant cells which it is desiredto transform direct with the gene to be transferred by introducing theforeign gene and plant protoplasts into a suitable solution and leavingthem therein until the foreign gene has been taken up by theprotoplasts. The transformation frequency can be increased by combiningthis step with techniques which are employed in microbiological researchfor gene transfer, for example by treatment with poly-L-ornithine orpoly-L-lysine, liposome fusion, DNA protein complexing, altering thecharge at the protoplast membrane, fusion with microbial protoplasts, orcalcium phosphate co-precipitation and, in particular, by treatment withpolyethylene glycol, heat shock and electroporation, as well as acombination of these last three mentioned techniques.

Suitable solutions into which the foreign gene and the receptorprotoplasts are introduced are preferably the osmotically stabilisedculture media employed for protoplast cultures.

Numerous culture media are already available which differ in theirindividual components or groups of components. However, the compositionof all media is in accordance with the following principle: they containa group of inorganic ions in the concentration range from about 10 mg/lto several hundred mg/l (so-called macroelements such as nitrate,phosphate, sulfate, potassium, magnesium, iron), a further group ofinorganic ions in maximum concentrations of several mg/l (so-calledmicroelements such as cobalt, zinc, copper, manganese), then a number ofvitamins (for example inositol, folic acid, thiamine), a source ofenergy and carbon, for example saccharose or glucose, and also growthregulators in the form of natural or synthetic phytohormones of theauxin and cytokinin classes in a concentration range from 0.01 to 10mg/l. The culture media are additionally stabilised osmotically withsugar alcohols (for example mannitol) or sugar (for example glucose) orsalt ions (for example CaCl₂), and are adjusted to a pH in the rangefrom 5.6 to 6.5.

A more detailed description of conventional culture media will be found,for example, in Koblitz, H., Methodische Aspekte der ZellundGewebezüchtung bei Gramineen unter besonderer Berücksichtigung derGetreide, Kulturpflanze XXII, 1974, 93-157.

A particularly suitable technique of gene transformation is“polyethylene glycol treatment”, where the term “polyethylene glycol”within the scope of this invention denotes not only the substancepolyethylene glycol itself, but will also be understood as generic termfor all substances that likewise modify the protoplast membrane and areemployed e.g. in the field of cell fusion. The term thus also comprisesother polyhydric alcohols of longer chain length, for examplepolypropylene glycol (425 to 4000 g/mole), polyvinyl alcohol orpolyhydric alcohols whose hydroxyl groups are partially or completelyetherified, as well as the detergents which are commonly employed inagriculture and tolerated by plants, and which are described e.g. in thefollowing publications:

“Mc Cutcheon's Detergents and Emulsifiers Annual” MC Publishing Corp.,Ridgewood, N.J., 1981;

Stache, H., “Tensid-Taschenbuch”, Carl Hanser Verlag, Munich/Vienna,1981

If polyethylene glycol itself is used (as in Examples 1 to 3, 5 and 7),then it is preferred to use a polyethylene glycol having a molecularweight in the range from 1000 to 10,000 g/mole, preferably from 3000 to8000 g/mole.

Of the above mentioned substances, it is preferred to use polyethyleneglycol itself.

A substantial and reproducible transformation frequency of 10⁻⁵ isachieved by means of the techniques described above. However, thisfrequency can be greatly improved on by the appropriate techniquesdescribed in more detail hereinafter.

In the polyethylene glycol treatment, the procedure can be for examplesuch that either a suspension of the protoplasts is added to a culturemedium and then the gene, which is normally employed as plasmid, isadded in a mixture of polyethylene glycol and culture medium, or,advantageously, protoplasts and gene (plasmid) are first added to theculture medium and then polyethylene glycol is added. In the process ofthis invention, electroporation and heat shock treatment have alsoproved particularly advantageous techniques.

In electroporation [Neumann, E. et al., The EMBO Journal 7, 841-845(1982)], protoplasts are transferred to an osmoticum, for example amannitol/magnesium solution and the protoplast suspension is introducedinto the electroporator chamber between two electrodes. By discharging acondenser over the suspension, the protoplasts are subjected to anelectrical impulse of high voltage and brief duration, thereby effectingpolarisation of the protoplast membrane and opening of the pores in themembrane.

In the heat treatment, protoplasts are suspended in an osmoticum, forexample a solution of mannitol/calcium chloride, and the suspension isheated in small containers, for example centrifuge tubes, preferably ina water bath. The duration of heating will depend on the chosentemperature. In general, the values are in the range of 40° C. for 1hour and 80° C. for 1 second. Optimum results are obtained at atemperature of 45° C. over 5 minutes. The suspension is subsequentlycooled to room temperature or lower.

It has also been found that the transformation frequency can beincreased by inactivating the extracellular nucleases. Such aninactivation can be effected by using divalent cations that aretolerated by plants, for example magnesium or calcium, and alsopreferably by carrying out the transformation at a high pH value, withthe optimum pH range being from 9 to 10.5.

Surprisingly, the selective use of these different methods results inthe enormous increase in transformation frequency that has long been anobjective in the field of genetic engineering.

The lower the transformation frequency in gene transformation, the moredifficult and time-consuming it is to find the few cloned cellsresulting from the transformed cells from among the enormous number ofnon-transformed clones. Where the transformation frequency is low, theuse of conventional screening techniques is almost or completelyimpossible, unless the gene employed is one with selective markerfunction (e.g. resistance to a specific substance). Low transformationfrequency thus requires a very substantial investment in time and effortwhen using genes without marker function.

In transformations using genes without marker function it is onlypossible to employ conventional screening techniques for finding clonedcells by selection efficiently and successfully if the transformationfrequency is in the order of percentages (about 10⁻²). As will be shownbelow, the desired transformation frequency can now be achieved by meansof the process of this invention. Surprisingly, the specific use ofdifferent techniques in the process of this invention results in anenormous increase in the previously attained transformation frequency upto 1 to 2%.

Combining foreign gene and receptor protoplasts before employing theother techniques such as polyethylene glycol treatment, electroporationand heat shock treatment brings about an improvement in transformationfrequency of the order of about a power of ten as compared with aprocedure in which the sequence of the steps employed is different.

Electroporation effects a 5- to 10-fold, and heat shock treatment a10-fold or greater, improvement in transformation frequency.

A combination of two or three of the following techniques has provedadvantageous: polyethylene glycol treatment, heat shock treatment andelectroporation, with particularly good results being obtained byemploying these techniques after the foreign gene and protoplasts havebeen introduced into a solution. The preferred technique is heat shocktreatment before the polyethylene glycol treatment and the optionalsubsequent electroporation. In general, the additional electroporationeffects a further increase in transformation frequency; but in somecases the results obtained by heat shock and polyethylene glycoltreatment are no longer essentially improved by additionalelectroporation.

Just as the techniques can be combined with one another, so it is alsopossible to combine the use of divalent cations which are tolerated byplants and/or carrying out the transformation at pH 9 to 10.5 both withindividual techniques, as well as combined techniques, preferably withpolyethylene glycol treatment, heat shock treatment and electroporation.The numerous combination possibilities permit the process of thisinvention to be adapted very well to the respective conditions.

The combination of heat shock treatment, polyethylene glycol treatmentand, optionally, electroporation subsequent to the already existingcombination of foreign gene and receptor protoplasts results in atransformation frequency of 10⁻² to 10⁻³.

Accordingly, the process of this invention permits a high transformationfrequency to be achieved without utilising biological vectors for thetransformation, for example cauliflower mosaic virus or Agrobacterium.

An advantageous method comprises for example transferring protoplasts toa mannitol solution and mixing the protoplast suspension so obtainedwith the aqueous solution of the gene. The protoplasts are thenincubated in this mixture for 5 minutes at 45° C. and subsequentlycooled to 0° C. over 10 seconds. Then polyethylene glycol (mol. wt. 3000to 8000) is added until the concentration is in the range from 1 to 25%,preferably about 8%. After cautious thorough mixing, treatment iscarried out in an electroporator. The protoplast suspension is thendiluted with culture medium and the protoplasts are taken into culture.

The process of this invention is suitable for the transformation of allplants, especially those of the systematic groups Angiospermae andGymnospermae.

Among the Gymnospermae, the plants of the Coniferae class are ofparticular interest.

Among the Angiospermae, plants of particular interest are, in additionto deciduous trees and shrubs, plants of the following families:Solanaceae, Cruciferae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae,Rutaceae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae andof the order Leguminosae, in particular of the family Papilionaceae.Preferred plants are representatives of the Solanaceae, Cruciferae andGramineae families.

To be particularly mentioned are plants of the species Nicotiana,Petunia, Hyoscyamus, Brassica und Lolium, as for example, Nicotianatabacum, Nicotiana plumbagenifolia, Petunia hybrida, Hyoscyamus muticus,Brassica napus, Brassica rapa and Lolium multiflorum.

In the field of transformation of plant cells, interest focuses inparticular on the high yield cultivated plants such as maize, rice,wheat, barley, rye, oats and millet.

All plants which can be produced by regeneration from protoplasts canalso be transformed utilising the process of this invention. So far ithas not been possible to manipulate genetically representatives of theGramineae family (grasses), which also comprises cereals. It has nowbeen shown that graminaceous cells, including cereal cells, can betransformed genetically by the above described method of direct genetransformation. In the same way, transformation of cultivated plants ofthe genus Solanum, Nicotiana, Brassica, Beta, Pisum, Phaseolus, Glycine,Helianthus, Allium, wheat, barley, oat, Setaria, rape, rice, Cydonia,Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis, Secale, Panicum,Saccharum, Coffea, Camellia, Musa, Ananas, Vitis or Citrus is possibleand desirable, even if the total yields and crop areas are smallerworldwide.

The proof of transformed genes can be adduced in a manner known per se,for example by crossing analyses and molecular biological assays,including in particular the Southern blot analysis and enzyme activitytests.

The Southern blot analysis can be carried out for example as follows:the DNA isolated from the transformed cells or protoplasts iselectrophoresed in 1% agarose gel after treatment with restrictionenzymes and transferred to a nitrocellulose membrane [Southern, E. M.,J. Mol. Biol. 98, 503-517 (1975)], and hybridised with the DNA whoseexistence it is desired to establish and which was nick-translated[Rigby, W. J., Dieckmann, M., Rhodes, C. and P. Berg, J. Mol. Biol. 113,237-51, (1977)] (DNA specific activity 5×10⁸ to 10×10⁸ c.p.m./μg). Thefilters are washed 3 times for 1 hour with an aqueous solution of 0.03 Msodium citrate and 0.3 M sodium chloride at 65° C. The hybridised DNA isvisualised by darkening an X-ray film for 24 to 48 hours.

Testing for enzyme activity—explained in more detail in the assay foraminoglycoside phosphotransferase (enzyme for kanamycin-specificphosphorylation)—can be carried out for example as follows: Callus orleaf pieces (100 to 200 mg) are homogenised in 20 μl of extractionbuffer in an Eppendorf centrifuge tube. The buffer is modified from thatused by Herrera-Estrella, L., DeBlock, M., Messens, E., Hernalsteens,J.-P., Van Montagu, M. and J. Schell, EMBO J. 2, 987-995 (1983) omittingbovine serum albumin and adding 0.1 M sucrose. The extracts arecentrifuged for 5 minutes at 12000 g and bromophenol blue is added tothe supernatant to a final concentration of 0.004%. The proteins in 35μl of supernatant are separated by electrophoresis in a 10%non-denaturing polyacrylamide gel. The gel is covered with a layer ofagarose gel containing kanamycin and γ-³²P labelled ATP, incubated, andthe phosphorylated reaction products are transferred to Whatman p81phosphocellulose paper. The paper is washed 6 times with deionised waterat 90° C. and then autoradiographed.

The following Examples illustrate the present invention in more detailbut without limiting the scope thereof. They describe the constructionof a hybrid gene and the insertion thereof in carrier DNA sequences ofcyclic character, the transfer of said hybrid gene into plant cells,selection of the transformed plant cells and regeneration of completeplants from the transformed plant cells as well as the genetic crossingand molecular biological analysis thereof.

In the Examples, the process of the invention is illustrated as follows:

1) by transformation of tobacco plants by transfer of the NPT II gene byjoining promoter and termination signals of the CaMV gene VI to the NPTII gene, inserting said gene into the pUC8 plasmid and transferring theresultant chimaeric plasmid into isolated tobacco protoplasts bypolyethylene glycol treatment;

2) by transformation of plants of the genus Brassica by transfer of theNPT II gene by joining promoter and termination signals of the CaMV geneVI to the NPT II gene, inserting this construction instead of the CaMVgene VI into the CaMV genome, and transferring the resultant chimaericplasmid into isolated Brassica protoplasts by polyethylene glycoltreatment, and

3) by transformation of plants of the genus Lolium by transfer of theNPT II gene by joining promoter and termination signals of the CaMV geneVI to the NPT II gene, inserting said gene into the pUC8 plasmid, andtransferring the resultant chimaeric plasmid into isolated Loliumprotoplasts by polyethylene glycol treatment.

Further, the advantageous effect on the transformation by heat shocktreatment and electroporation as well as the combined method of heatshock treatment, polyethylene glycol treatment and electroporation aftercombining protoplasts and NPT II gene will be exemplified.

EXAMPLE 1 Transformation of Cells of Nicotiana tabacum c.v. Petit HavanaSRI by Transfer of the NPT II Gene

a) Construction of the pABDI Plasmid

The freely available plasmids pKm 21 and pKm 244 [Beck, E. et al., Gene19, 327-336 (1982)] are cut with the PstI restriction endonuclease. Thefragments of the plasmids which are used for recombination are purifiedby electrophoresis in 0.8% agarose gel. The plasmid pKm 21244 resultingfrom the combination of the fragments contains a combination of the 5′-and 3′-Bal 31 deletions of the NPT II gene, as described by Beck et alin Gene 19, 327-336 (1982). Joining the promoter signal of cauliflowermosaic virus to the HindIII fragment of the plasmid pKm 21244 iseffected by constructing the linker plasmid pJPAX. The coupling plasmidpJPAX is obtained from the plasmids pUC8 and pUC9 [Messing, J. and J.Vieira, Gene 119, 269-276 (1982)]. 10 base pairs of the linker sequenceof the plasmid pUC9 are deleted by restriction at the HindIII and SalIsites and the resultant cohesive ends are filled in by treatment withthe polymerase I Klenow fragment [Jacobson, H. et al., Eur. J. Biochem.45, 623, (1974)] and ligating the polynucleotide chain, thus restoringthe HindIII site. An 8 base pair synthetic XhoI linker is inserted atthe SmaI site of this deleted linker sequence. Recombination of theappropriate XorI and HindIII fragments of the plasmid pUC8 and of themodified plasmid pUC9 yields the plasmid pJPAX with a partiallyasymmetric linker sequence containing the following sequence ofrestriction sites: EcoRI, SMaI, BamHI, SalI, PstI, HindIII, BamHI, XhoIand EcoRI. Joining of the 5′ expression signals of the CaMV gene VI andthe HindIII fragment of the NPT II gene is carried out on the plasmidpJPAX by inserting the promoter region of the CaMV VI gene between thePstI and HindIII sites. The plasmid so obtained is restricted at itssingle HindIII site and the HindIII fragment of the plasmid pKm 21244 isinserted into this restriction site in both orientations, yielding theplasmids pJPAX CaKm⁺ and pJPAX CaKm⁻. To provide an EcoRV site near the3′-terminal region of the NPT II hybrid gene, a BamHI fragment of theplasmid pJPAX CaKm⁺ is inserted into the BamHI site of the plasmid pBR327 [Soberon, X. et al., Gene 9, 287-305 (1980)], yielding the plasmidpBR 327 CaKm. The EcoRV fragment of the plasmid pBR 327 CaKm, whichcontains the new DNA construction, is used to replace the EcoRV regionof the CaMV gene VI, which is cloned at the SalI site in the plasmidpUC8, thereby placing the protein-coding DNA sequence of the NPT II geneunder the control of the 5′ and 3′ expression signals of the cauliflowermosaic gene VI. The plasmids so obtained are designated pABDI and pABDIIrespectively (q.v. FIG. 1).

b) Transformation of Protoplasts of Nicotiana tabacum c.v. Petit HavanaSRI by Transfer of the NPT Gene as Part of the Plasmid pABDI by PEGTreatment

Tobacco protoplasts at a population density of 2·10⁶ per ml aresuspended in 1 ml of K₃ medium [q.v. Z. Pflanzenphysiologie 78, 453-455(1976); Mutation Research 81 (1981) 165-175], containing 0.1 mg/l of2,4-dichlorophenoxyacetic acid, 1.0 mg/l of 1-naphthylacetic acid and0.2 mg/A of 6-benzylaminopurine, which protoplasts have been obtainedbeforehand from an enzyme suspension by flotation on 0.6 molar sucroseat pH 5.8 and subsequent sedimentation (100 g for 5 minutes) in 0.17 Mcalcium chloride at pH 5.8. To this suspension are added, in succession,0.5 ml of 40% polyethylene glycol (PEG) with a molecular weight of 6000in modified (adjusted again to pH 5.8 after autoclaving) F-medium[Nature 296, 72-74 (1982)] and 65 μl of an aqueous solution containing15 μg of the plasmid pABDI and 50 μg of calf thymus DNA. This mixture iscultured for 30 minutes at 26° C. with occasional agitation andsubsequent stepwise dilution with F medium. The protoplasts are isolatedby centrifuging (5 minutes at 100 g) and resuspended in 30 ml of freshK₃ medium. Further incubation is carried out in 10 ml portions in Petridishes of 10 cm diameter at 24° C. and in the dark. The populationdensity is 6.3·10⁴ protoplasts per ml. After 3 days the culture mediumin each dish is diluted with 0.3 parts by volume of fresh K₃ medium andincubated for a further 4 days at 24° C. and 3000 lux. After a total of7 days, the clones developed from the protoplasts are embedded in aculture medium solidified with 1% of agarose and containing 50 mg/l, ofkanamycin, and cultured at 24° C. in the dark by the bead type culturemethod [Plant Cell Reports, 2, 244-247 (1983)]. The culture medium isreplaced every 5 days by fresh nutrient solution of the same kind.

c) Regeneration of Kanamycin-Resistant Tobacco Plants

After 3 to 4 weeks of continued culturing in kanamycin-containingculture medium, the resistant calli of 2 to 3 mm diameter aretransferred to agar-solidified LS culture medium [Physiol. Plant 18,100-127 (1965)], containing 0.05 mg/l of 2,4-dichlorophenoxyacetic acid,2 mg/l of 1-naphthylacetic acid, 0.1 mg/l of 6-benzylaminopurine, 0.1mg/l of kinetin and 75 mg/l of kanamycin. Kanamycin-resistant Nicotianatabacum Petit Havana SRI plants are obtained by inducing shoots on LSmedium containing 150 mg/l of kanamycin and 0.2 mg/l of6-benzylaminopurine, and subsequent rooting on T medium [Science 163,85-87 (1969)].

d) Detection of the NPT II Gene in Hereditary Material of Plants

Samples of 0.5 g of callus of the transformed cell cultures or leaftissue of the plants regenerated therefrom are homogenised at 0° C. in15% saccharose solution containing 50 mmol/l of 1-ethylenediamineN,N,N′,N′-tetraacetic acid (EDTA), 0.25 mol/l of sodium chloride and 50mmol/l of α,α,α-tris(hydroxymethyl)methylamine hydrochloride (TRIS-HCl)at pH 8. Centrifugation of the homogenate for 5 minutes at 1000 g givesa crude nuclear pellet which is resuspended at pH 8.0 in 15% saccharosesolution containing 50 mmol/l of EDTA and 50 mmol/l of TRIS-HCl. Sodiumdodecyl sulfate is added to a final concentration of 0.2% and heated for10 minutes to 70° C. After cooling to 20°-25° C., potasasium acetate isadded to the mixture to a concentration of 0.5 mol/l This mixture isincubated for 1 hour at 0° C. The precipitate is centrifuged for 15minutes at 4° C. in a microcentrifuge. The DNA is precipitated from thesupernatant with 2.5 volumes of ethanol at 20°-25° C. The isolated DNAis dissolved in a solution of 10 mmol of TRIS-HCl containing 10 μg/ml ofribonuclease A. After incubation for 10 minutes at 37° C., proteinase Kis added to a concentration of 250 250 μg/ml and incubation is continuedfor 1 hour at 37° C. The proteinase K is removed by phenol andchloroform/isoamyl alcohol extractions. The DNA is precipitated from theaqueous phase by addition of 0.6 part by volume of a 0.6 molar solutionof sodium acetate in isopropanol and dissolved in 50 μl of a solutioncontaining 10 mmol/l of TRIS-HCl and 5 mmol/l of EDTA at pH 7.5. Thispreparation yields DNA sequences which contain substantially more than50,000 base pairs. Restriction of this DNA with EcoRV endonuclease,hybridisation of the fragments with radioactively labelled HindIIIfragments of the NPT II gene and comparison with the plasmid pABDI show,in Southern blot analysis, the presence of the NPT II gene in the cellnucleus DNA of the transformed Nicotiana tabacum cells.

e) Evidence of the Transfer of the Transformed Gene to Sexual Offspringand of its Heredity as Normal Plant Gene

Extensive genetic crossing analyses and detailed molecular biologicalstudies (for example Southern blot analysis of the DNA of the plantgenome; investigation of the enzyme activity of the aminoglycosidephosphotransferase, i.e. the enzyme for the kanamycin-specificphosphorylation) with the genetically transformed plants (firstgeneration and progeny) have yielded the following results:

1. the baceterial gene is stably integrated into the plant genome;

2. it is normally unchanged and regularly transferred to crossedprogeny;

3. its heredity corresponds to that of a natural, simple dominant plantgene;

4. the molecular analysis by DNA hybridisation and enzyme test confirmsthe results of the genetic crossing analysis;

5. the genetically transformed plants retain their normal, naturalphenotype during the treatment, i.e. no undesirable modifications areobserved.

These results show that the process of this invention for the directtransfer of a gene into protoplasts affords the best mode ofspecifically transforming plant material genetically. The genetictransformation is stable and unwanted modifications in the genotype ofthe plant do not occur. Parallel results are also obtained when carryingout the transformation described in the foregoing Example with Nicotianaplumbagenifolia, Petunia hybrida, Hyoscyamus muticus and Brassica napus.

EXAMPLE 2 Transformation of Cells of Brassica rapa c.v. Just Right byTransfer of the NPT II Gene

a) Construction of the Plasmid pCaMV6Km

The plasmid pBR 327 CaKm⁺ described in Example 1a is digested withrestriction endonuclease EcoRV and the EcoRV restriction fragmentcontaining the kanamycin-resistant gene (NPT II) is used to replace theEcoRV fragment of the plasmid pCa20-Bal I, which fragment contains thegene VI of cauliflower mosaic virus, yielding the plasmid pCaMV6Km (FIG.2). The plasmid Ca20-Bal I is a chimaeric CaMV plasmid which is derivedfrom the natural deletion mutant CN4-184. The entire region II ismissing from this plasmid, except for the first 5 codons and thetranslation stop signal TGA. An XhoI coupling component was insertedimmediately before the stop codon in region II.

b) Transformation of Protoplasts of Brassica rapa c.v. Just Right byTransfer of the NPT Gene as Part of the Plasmid pCaMV6Km by PEGTreatment

Brassica rapa protoplasts are washed with a suitable osmoticum andsuspended in a population density of 5·10⁶ per ml in a culture mediumprepared according to Protoplasts 83, Proceedings ExperientiaSupplementum, Birkhäuser Verlag, Basel, Vol. 45 (1983), 44-45. 40%polyethylene glycol (PEG) with a molecular weight of 6000, dissolved inmodified F medium (pH 5.8) (q.v. Example 1b), is mixed with theprotoplast suspension to a final concentration of 13% PEG. To thismixture is added immediately a solution of 10 μg of plasmid pCaMV6Kmdigested with endonuclease SalI, and 50 μg of calf thymus DNA in 60 μgof water. With occasional agitation, the mixture is incubated for 30minutes at 20°-25° C. Then 3×2 ml of modified F medium (6 ml in all) and2×2 ml of culture medium (4 ml in all) are added at 5 minute intervals.The protoplast suspension is transferred to 10 cm Petri dishes and madeup to a total volume of 20 ml with additional culture medium. Theseprotoplast suspensions are incubated in the dark for 45 minutes at 26°C. The protoplasts are isolated by sedimentation for 5 minutes at 100 g,taken up in an initially liquid and then solidifying agarose gel culturemedium and cultured by the bead type culture method [Plant Cell Reports2, 244-247 (1983)]. After 4 days, in the development stage of the firstcell division, kanamycin is added to the cultures in a concentration of50 mg/l. The liquid culture medium surrounding the agarose segments isreplaced every 4 days by fresh kanamycin-containing nutrient solution.After 4 weeks the kanamycin-resistant clones are isolated and thenfurther cultured by providing them weekly with kanamycin-containingnutrient solution (50 mg/l).

c) Detection of the NPT II Gene in the Hereditary Material of the Plants

The presence of the NPT II gene in the cell nucleus of the transformedBrassica rapa cells can be detected by isolation of the cell nucleus DNAand restriction thereof and hybridisation of the DNA fragments asdescribed in Example 1 d).

EXAMPLE 3 Transformation of Protoplasts of Graminaceous Plants of theSpecies Lolium multiflorum

Protoplasts of Lolium multiflorum (Italian ryegrass) are taken up at aconcentration of 2·10⁶ per ml in 1 ml of 0.4 molar mannitol at pH 5.8.To this suspension are added, in succession, 0.5 ml of 40% polyethyleneglycol (PEG) with a molecular weight of 6000 in modified (pH 5.8) Fmedium [Nature 296, 72-74 (1982)], and 65 μl of an aqueous solutioncontaining 15 μg of the plasmid pABDI and 50 μg of calf thymus DNA. Thismixture is incubated for 30 minutes at 26° C. with occasional agitationand subsequently diluted with F medium, as described in Nature 296(1982), 72-74. The protoplasts are isolated by centrifugation (5 minutesat 100 g) and taken up in 4 ml of CC culture medium [Potrykus, Harms,Lörz, Callus formation from cell culture protoplasts of corn (Zea MaysL.), Theor. Appl. Genet. 54, 209-214 (1979)] and incubated in the darkat 24° C. After 14 days the developing cell cultures are transferred tothe same culture medium, but with the antibiotic G-418 (commerciallyavailable; GIBCO EUROPE Product Catalogue, Catalogue No. 0661811). G-418is toxic to Lolium cells at a concentration of 25 mg/l and permitssolely the further development of cells which have taken up thebacterial gene for kanamycin resistance. G-418 is a kanamycin analogwith substantially better activity in graminaceous cells than kanamycinitself. Resistant cell colonies are transferred to agar medium (the samemedium as above, 25 ml/l G-418, without osmoticum) and, after reaching asize of several grams fresh weight per cell colony, analysed for thepresence of the bacterial gene and for the biological activity of thegene. The former analysis is made by hybridisation of a radioactivelylabelled DNA sample of the gene with DNA which has been isolated fromthe cell culture; while the latter is made by detecting the enzymeactivity by phosphorylation of kanamycin with radioactive ATP. Bothmolecular analyses yielded unequivocal proof of the genetictransformation of the cell colonies which had been selected on G-418.The assays constitute the first proof of the genetic transformation ofprotoplasts of graminaceous plants and furthermore prove that, inprinciple, protoplasts of grasses can be genetically manipulated by thedescribed process. The possibility of genetically manipulatingcultivated grasses, for example cererals, is thus also afforded.

EXAMPLE 4 Transformation of Cell Culture Cells of Nicotiana tabacum byTransferring the NPT Gene by Means of Electroporation

Protoplasts are produced by sedimentation from 50 ml of a log phasesuspension culture of the nitrate reductase deficiency variant ofNicotiana tabacum, cell strain nia-115 [Müller, A. J. and R. Grafe, Mol.Gen. Genet. 161, 67-76 (1978)], and resuspended in 20 ml of enzymesolution [2% Cellulase Onozuka R-10, 1% Mazerozym R-10 and 0.5%Driselase (available from Chemische Fabrik Schweizerhalle, Basel) in awash solution (0.3 M mannitol, 0.04 M calcium chloride and 0.5%2-(N-morpholino)ethanesulfonic acid), adjusted to pH 5.6 with KOH] andincubated for 3 hours on a gyratory shaker at 24° C. The protoplasts arethen separated from undigested tissue by filtering them through a 100 μmmesh sieve. An equal volume of 0.6 M sucrose is added and the suspensionis centrifuged for 10 minutes at 100 g. The protoplasts floating on thesurface are collected and washed 3 times by sedimentation in the washsolution.

Transformation is carried out by electroporation. The chamber of aDialog® “Porator” (available from Dialog GmbH, Harffstr. 34, 4000Düsseldorf, West Germany) is sterilised by washing with 70% ethanol andthen 100% ethanol and dried by a current of sterile air from aventilator with laminary air flow). The protoplasts are suspended at aconcentration of 1×10⁶/ml in 0.4 M mannitol solution, adjusted withmagnesium chloride to a resistance of 1.4 kOhm and pABDI DNA is added ina concentration of 10 μg/ml. 0.38 ml samples of this protoplastsuspension are subjected 3 times at 10 second intervals to a charge of1000 volts or to a charge, of 2000 volts. The protoplasts are thencultured in a concentration of 1×10⁵/ml in 3 ml of AA-CH medium [AAmedium of Glimelius, K. et al., Physiol. Plant. 44, 273-277 (1978)],modified by increasing the inositol concentration to 100 mg/l and thesaccharose concentration to 34 g/l, as well as by adding 0.05 ml/l of2-(3-methyl-2-butenyl)adenine, and which is solidified by a 0.6% contentof agarose (Sea Plaque, FMC Corp., Marine Colloids Division, P.O. Box308, Rockland, Me. 04841, USA). After 1 week, the agarose layercontaining the protoplasts is transferred to 30 ml of liquid AA-CHmedium which contains 50 mg/l of kanamycin. After 3 weeks, during whichtime half the liquid medium is replaced weekly by fresh medium of thesame composition, the transformed cell colonies can be observedvisually. Four weeks after being transferred to the medium containingkanamycin, these cell colonies are transferred to AA medium (Glimelius,K. et al., Physiol. Plant. 44, 273-277 (1978); 0.8% agar), whichcontains 50 mg/l of kanamycin, for further culturing and investigation.Confirmation of the successful transformation is by DNA hybridisationand testing for the enzyme activity of aminoglycosidephosphotransferase.

Analogous assays with protoplasts of Brassica rapa and Loliummultiflorum also result in successful transformations.

EXAMPLE 5 Transformation of Cells of Nicotiana tabacum by Transfer ofthe NPT II Gene by Electroporation

The preparation of the electroporator is as described in Example 4 andof the protoplasts as in Example 1.

For transformation, protoplasts of Nicotiana tabacum are resuspended ina concentration of 1.6×10⁶/ml in mannitol solution (0.4 M, buffered with0.5% w/v of 2-(N-morpholino)ethanesulfonic acid; pH pH 5.6). Theresistance of the protoplast suspension is measured in the poratorchamber (0.38 ml) and adjusted to 1 to 1.2 kOhm with magnesium chloridesolution (0.3 M). 0.5 ml samples are put into capped plastic tubes (5 mlvolume) to each of which are added initially 40 μl of water containing 8μg of pABDI (linearised with SmaI) and 20 μg of calf thymus DNA, andthen 0.25 ml of polyethylene glycol solution (24% w/v in 0.4 Mmannitol). Nine minutes after addition of the DNA, 0.38 ml portions areput into the pulse chamber and 10 minutes after the addition of DNA, theprotoplast suspensions present in the chamber are subjected to 3impulses (1000-2000 volts) at 10 second intervals. The treated portionsare put into Petri dishes of 6 cm diameter and kept for 10 minutes at20° C. Then 3 ml of K₃ medium containing 0.7% w/v of Sea Plaque agaroseare added to each Petri dish and the contents of the dish are thoroughlymixed. After solidification of the contents of each dish, the culturesare kept for 1 day at 24° C. in the dark and then for 6 days in light.The protoplast-containing agarose is then cut into quarters andintroduced into liquid medium. The protoplasts are then cultured by thebead type culturing method. Callus tissues which are obtained byselection of the transformed material with kanamycin and plantsregenerated therefrom contain the NPT II enzyme (aminoglycosidephosphotransferase) as product of the NPT II gene.

Electroporation induces a 5- to 10-fold increase in the frequency oftransformation compared with the method without electroporation.Analogous assays with Brassica rapa c.v. Just Right and Loliummultiflorum also bring about an increase in the frequency oftransformation of the same order of magnitude.

EXAMPLE 6 Transformation of Cells of Nicotiana tabacum by Transfer ofthe NPT II Gene by Means of Heat Shock

Protoplasts isolated from leaves or cell cultures of Nicotiana tabacumare isolated as described in Examples 1 and 4 and transferred to anosmotic medium as described in the preceding Examples. The protoplastsuspensions are kept for 5 minutes at 45° C., cooled with ice for 10seconds and then the plasmid pABDI is added as described in Examples 1and 4. The heat shock treatment increases the transformation frequencyby a factor of 10 or higher compared with a transformation carried outwithout this treatment.

Analogous assays with the protoplasts and plasmids described in Examples2 and 3 also bring about an increase in the frequency of transformationof the same order of magnitude.

EXAMPLE 7 Transformation of Different Plant Cells by Transfer of the NPTII Gene by Combining Protoplasts and Gene as First Step and SubsequentCombined Treatment

Protoplasts of the plants:

Nicotiana tabacum c.v. Petit Havana SRI (A),

Brassica rapa c.v. Just Right (B) and

Lolium multiflorum (C)

are isolated and transferred to an osmotic medium as described inExample 5. The protoplast suspensions of A) and C) are mixed with theplasmid pABDI (Example 1a), and those of B) with the plasmid pCaMV6Km(Example 2a) as described in Examples 1 to 3, but without simultaneoustreatment with polyethylene glycol. The protoplast suspensions are thensubjected to a heat shock treatment as described in Example 6, then to apolyethylene glycol treatment as described in Examples 1 to 3, andfinally subjected to electroporation as described in Example 5. Thetransformation frequency in this procedure is in the range from 10⁻³ to10⁻², but may be from 1 to 2% depending on the conditions. (Thetransformation frequency in Examples 1 to 3 is in the order of about10⁻⁵). Results in the range from 10⁻³ to 10⁻² are also obtained if,after combining protoplasts and plasmids, the subsequent steps of heatshock, polyethylene glycol treatment and electroporation are employed indifferent sequence.

What is claimed is:
 1. A transgenic Brassica cell comprising foreign DNA unaccompanied by T-DNA border regions of a Ti plasmid, said foreign DNA being under the control of plant expression signals and integrated into the plant hereditary material, such that said foreign DNA is transmissible to the progeny of a plant regenerated from said transgenic plant cell, wherein said transgenic plant cell is transformed by a process for the stable integration of said foreign DNA comprising: contacting said foreign DNA directly with a plant protoplast in a medium under conditions that render the plant protoplast membrane permeable to DNA molecules until said foreign DNA has been taken up by the protoplast, wherein said stable integration is carried out by a technique selected from the group consisting of polyethylene glycol treatment, electroporation, heat shock treatment, and combinations thereof.
 2. A transgenic Brassica plant cell according to claim 1 wherein the plant cell is selected from the group consisting of Brassica napus and Brassica rapa plant cells.
 3. A transformed living Brassica protoplast or plant cell, derived from the transgenic Brassica plant cell of claim 1, each comprising said foreign DNA.
 4. A transformed living Brassica protoplast, cell, tissue or whole plant, derived from the transgenic Brassica cell of claim 1, each comprising said foreign DNA.
 5. A transgenic Brassica plant derived from the Brassica cell of claim 1, said Brassica plant comprising said foreign DNA.
 6. The transgenic Brassica plant of claim 5, wherein said foreign DNA comprises a structural gene flanked by plant expression signals.
 7. The transgenic Brassica plant of claim 5, wherein said foreign DNA comprises a gene whose structural part is of plant, animal, microbial, viral or synthetic origin and whose expression signals are of plant, animal or synthetic origin.
 8. The transgenic Brassica plant of claim 5, wherein said Brassica is selected from the group consisting of Brassica napus and Brassica rapa.
 9. The transgenic Brassica plant of claim 6, wherein said Brassica is selected from the group consisting of Brassica napus and Brassica rapa.
 10. The transgenic Brassica plant of claim 7, wherein said Brassica is selected from the group consisting of Brassica napus and Brassica rapa. 